The reversible phosphorylation of proteins on serine and threonine residues plays a key regulatory role in the transduction of signals that mediate cellular responses to extracellular cues. To maintain cellular homeostasis, the activities of the kinases and phosphatases that catalyze protein phosphorylation and dephosphorylation, respectively, must be tightly controlled in vivo. Aberrant regulation of these enzymes has been linked with various pathological conditions such as cancer, diabetes, immune system disorders, and neurodegenerative diseases. A growing body of evidence indicates that regulation of kinases and phosphatases involves their association with specific cellular proteins in multiprotein complexes. Published findings from the applicant's laboratory have revealed that specific protein serine/threonine phosphatase 2A (PP2A) holoenzymes form individual macromolecular complexes with multiple protein kinases that include Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) and the mitogen-activated protein kinase kinase kinase Raf1 (J. Biol. Chem. 280:42644, 2005;J. Biol. Chem. 280:35974, 2005;J. Biol. Chem. 274:687, 1999;Science 280:1258, 1998). Within the CaMKIVPP2A and Raf1PP2A complexes, the associated PP2A negatively and positively regulates CaMKIV and Raf1 activation, respectively. The overall objective of this proposal is to understand the structure, function, and regulation of CaMKIVPP2A and Raf1PP2A complexes. The applicant hypothesizes that these signaling complexes play an essential role in the control of mitogenic, differentiative, and apoptotic responses. To address this central hypothesis, and to further explore the structure and regulation of these signaling modules, the applicant proposes a series of complementary biochemical, immunological, and genetic investigations that will uncover i) the structural nature of the kinase-PP2A interaction and the precise protein-protein interaction domains, ii) the role of CaMKIVPP2A and Raf1PP2A complexes in cell growth, differentiation, and survival, and iii) the regulatory mechanisms controlling kinasePP2A signaling complexes in response to various cell stimuli and mediators of cell stress. These studies will provide fundamental mechanistic insights into the molecular biology of CaMKIVPP2A and Raf1PP2A signaling modules that will undoubtedly be applicable to other kinasePP2A complexes. Given the importance of Raf1, CaMKIV, and PP2A in cell growth, differentiation, and proliferation, the proposed studies may also reveal novel therapeutic targets for pathophysiological processes underlying oncogenesis and neurodegeneration.